What is a Nitric Oxide Synthase?

Nitric Oxide Synthase is a heme protein similar to cytochrome P4501

There are several forms of nitric oxide synthase all of which operate in different parts of the body.  These isoforms are; neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and bacterial nitric oxide synthase (bNOS)2. The neuronal, inducible, and endothelial isoforms can be found in humans while the bacterial isoform is found in bacteria. A single copy for each of the three distinct genes for the human isoforms are found in the haploid human genome. The NOS genes suggest a common ancestral NOS gene due to similar genomic structure. NOSs have a bidomain stucture, where an N-terminal oxygenase domain is linked by a CaM-recognition site to a C-terminal reductase domain.6


Fig. 1

Nitric oxide synthase complexed with arginine from Geobacillus Stearothermophilus from JMol.
Nitric oxide synthases are responsible for the synthesis of nitric oxide from L-arginine2.  In order for this two step heme-based oxygenation to occur, several cofactors are required.  All three major isoforms in humans have a carboxyl terminated reductase end which binds flavins (FMN and FAD) as well as NADPH2.  They also possess a amino terminal oxygenase which contains the heme binding domain.  This oxygenase is also where the cofactors H4B (tetrahydrobiopterin) binds as well as the Arginine needed for the reaction.  These two domains are linked by a calmodulin binding domain which acts as a switch for the enzyme3.

It is very easy to say that all NOS's have the same mechanism and therefore they are the same, however all three isoforms have minor structural differences which separate them.  One of these major differences is there is a difference in amino acids in the active site and hydrogen bond with the substrate giving the enzyme it's activity. A lot of these differences are discovered by studying inhibitors of the enzymes.  Flinspach et. al. studied selective dipeptide inhibitors for one isoform over another.  From this study it was discovered that a difference in the active site between eNOS and nNOS allwed these inhibitors to be selective for one isoform over another.  This difference was the amino acid composition in the active site of the enzymes.  It was determined that the selectivity of the inhibitors occurred near the alpha-amino group of the inhibitor.  In eNOS this position is occupied by Asn368 in eNOS and Asp597 in nNOS7.

Fig. 2

Dipeptide inhibitor bound to nNOS (a) and eNOS (b), each isoform adopting a different conformation7.

Fig. 3

Overlay of the Binding of inhibitor by iNOS (yellow) and nNOS (black)8.
This single difference in amino acid identitiy leads to a completely different conformation when the inhibitor is bound which provides selectivity between the isoforms of NOS.

Another structural difference is between iNOS and both eNOS and nNOS.  iNOS possesses a Asn368 residue in place of a Ser residue in both eNOs and nNOS.  "Although the side chain of either Asn or Ser forms a hydrogen bond to a conserved His residue from the nearby helix H7, the bulkier Asn in iNOS forces the backbone of the S15 strand to move into the active site by as much as 1.1 A ˚ at residue Gly365 compared to the strand position in nNOS or eNOS8"
Nitric oxide synthase is a very important enzyme in the human body because it synthesizes nitric oxide (NO) from L-Arginine2.


Scheme 1

Overall reaction catalysed and cofactors of NOS6

Electrons are donated by NADPH to the reductase domain of the enzyme and proceed via FAD and FMN redox carriers to the oxygenase domain. In the oxygenase domain, they interact with the haem iron and BH4 at the active site to catalyse the reaction of oxygen with L-arginine, generating citrulline and NO as products. Electron flow through the reductase domain requires the prescence of bound Ca2+/CaM.6

Fig. 4

The oxidoreductive cycle of NOS converting L-Arg to the L-NHA intermediate8.
The catalytic cycle of NOS is similar to that of cytochrome P450 involving a 2 electron reduction from the reductase domain

 to the heme domain in two steps8.  In one of these steps (unlike cytochrome P450) an electron from another reduction 
molecule (H4B) is used8.  The oxidation-reduction of the iron during catalysis can be seen in figure 4. "One electron from the flavin reductase domain reduces the resting state, Fe(III)/H4B, to

Fe(II)/H4B. The ferrous heme binds O2 to give Fe(II)–Oor Fe(III)–O.2-/H4B, then the iron-bound dioxygen is reduced by one electron from H4B forming Fe(III)–O.2-/H4B. Two protons are generally considered to be needed for cleavage of the O–O bond in order to generate the reactive Fe(III)–O species and release H2O. The iron-bound oxygen inserts into the N–H bond of a terminal guanidino nitrogen in L-Arg forming L-NHA and Fe(III)/H3B.. The H3B. is reduced back to the resting state, Fe(III)/H4B, by an electron from the flavins in the reductase domain8". The full reaction scheme can be seen below.

Scheme 2

Mechanistic Speculation on the formation of NO and citrulline from L-Arginine4.


Function-Diseases Caused by NOS Malfunction

Fig. 5

This is an important reaction because NO has many biological functions in the human body.  These functions include but are not limited to regulation of blood flow, neurotransmission, mediation of immunoresponse, mediated cytotoxicity in microbes and tumor cells, and a role in sexual function1,2.  This enzyme is also important because if it is not functioning properly it can cause several serious diseases such as stroke, septic shock, and Alzheimer's disease5.  These are all caused by the over or under production of NO by this enzyme1,2,9.  It has also been shown that "the expression of NOS isoforms and activity seem to be cell-specific9".  

Fig. 6

Immunohitochemistry of Liver Cells demonstrating the presence of NOS (red, brown) in different cells during disease. Endolthelium (black), Hepatocytes (white), and Kupffer Cells (Gray)9
Many diseases caused by malfunctions of NOS isoforms can be attributed to compartmentalization of the isforms.  For example, Villaneuva and Giulivi determined that even though NO is made in a certain locatation within a cell, it's diffusion coefficient and half life allow it to travel to the entire organ9.  This could lead to many diseases such as Diabetes.  It is displayed immunohistochemistry that in lung liver cells of patients with diabetes, the amount of eNOS in the cells is higher than normal.  This is the same for patients experiencing endotoxic shock9.  The increase in NOS results in an increase in NO in these cells which is believed to be part of the cause of the diseases.

Another major disease which NOS has been linked to is cancer.  It has been shown that NOS plays an important role in the biology of many forms of tumor cells10.  It was shown that NO synthase has higher activity in invasive tumor cells than in normal breast tissue as well as benign tumor cells.  This could be a link for treatment of breast cancer if new was to inhibit NOS i deduced.

Fig. 8

Measure of nitrite and nitrate production in tumor cells and the measure of NOS activity in the same tumor cells10.
Another major disease which has been linked to NOS is heart disease.  It has been shown in many papers that limited bioavailability of NO can lead to atherosclerosis which is the accumulation of fatty materials along the walls of the arteries.  In this study by Schade et. al., attempts are being made to increase the bioavailabilty of NOS (increasing amount of NO) in endothelial tissues leading to a decrease in heart disease11.  Several of the treatments to induce NO production in the endothelial tissue and treat heart disease is to supplement with L-arginine causeing eNOS to produce more NO, regulate arginases and nitric oxide synthases, and H4B supplementation11.  These methods have been proven to treat cardiovascular disease, however more research is needed to produce a time dependent solution that releases NO exactly when it is needed11.

Fig. 8

Regulatory points for supplementation of NOS.  This is the most effective treatment for cardiovascular disease11.

Nitric oxide synthase has been linked to many specific diseases which could lead to new forms of treatment can be developed involving selective inhibition of these enzymes.

Current Studies
Nitric oxide synthase is an important enzyme because of all of the functions NO has within the human body.  Even though the function and active site of this enzyme is well understood there have been some discrepancies among scientists regarding some of the functions of NO.  For example, Alderton et. al brings up a point that it is of great debate whether L-citrulline is produced by NOS or if it is a mix of L/D-citruline12.  It is also mentioned that researched have been discussing whether NO is made directly by NOS or if a nitroxyl (NO-) ion or peroxynitrite (ONOO-) is formed first and then through subsequent reactions within the cell these ions are converted to NO12.  Knowing the reaction products will help scientists fully understand the reaction mechanism of NOS but right now it is a controversies among researchers.

Another controversy concerning NOS was brought up by Grisham et. al. who is describing the role of iNOS in human inflammatory bowel disease (Crohn's Disease, uncrative colitis)13.  It is stated that in several experiments it has been shown that NO produced by iNOS plays a role in the pathophysiology of the disease, but in others, it has been shown that NO from iNOS has no role in the disease or may even act to protect some of the inflammatory infected tissue13.

  Even though a lot about NOS is now understood there are still some questions and controversies regarding the mechanism as well as the role of NO produced by these enzymes within the human body.